Characterization of the ospZ Promoter in Shigella flexneri and Its Regulation by VirB and H-NS

Abstract

cOspZ is an effector protein of the type III secretion system in Shigella spp. that downregulates the human inflammatory response during bacterial infection. The ospZ gene is located on the large virulence plasmid of Shigella. Many genes on this plasmid are transcriptionally repressed by the nucleoid structuring protein H-NS and derepressed by VirB, a DNA-binding protein that displays homology to the plasmid partitioning proteins ParB and SopB. In this study, we characterized the ospZ promoter and investigated its regulation by H-NS and VirB in Shigella flexneri. We show that H-NS represses and VirB partially derepresses the ospZ promoter. H-NS-mediated repression requires sequences located between ؊731 and ؊412 relative to the beginning of the ospZ gene. Notably, the VirB-dependent derepression of ospZ requires the same VirB binding sites as are required for the VirBdependent derepression of the divergent icsP gene. These sites are centered 425 bp upstream of the ospZ gene but over 1 kb upstream of the icsP transcription start site. Although these VirB binding sites lie closer to ospZ than icsP, the VirB-dependent increase in ospZ promoter activity is lower than that observed at the icsP promoter. This indicates that the proximity of VirB binding sites to Shigella promoters does not necessarily correlate with the level of VirB-dependent derepression. These findings have implications for virulence gene regulation in Shigella and other pathogens that control gene expression using mechanisms of transcriptional repression and derepression.

H-NS, Its Family Members and Their Regulation of Virulence Genes in Shigella Species

“…VirB up-regulates greater than thirty virulence plasmid genes [48,49,50,51,52,53,54], including those found within the 30 kb ipa-mxi-spa region, which encode structural components of the type III secretion system and their effectors, the MxiE transcriptional regulator (Figure 2) as well as several other genes outside of this region. The activity of VirB as a transcriptional anti-silencing protein is demonstrated by work at the icsB , icsP and ospZ promoters, where VirB has no effect on promoter activity in the absence of functional H-NS [34,50,54]. If VirB solely functions to relieve transcriptional silencing by H-NS, as predicted by the experiments described above, then any gene found to be directly regulated by VirB will be silenced by H-NS.…”

Characterization of SlyA in Shigella flexneri Identifies a Novel Role in Virulence

“…To determine if SlyA-dependent upregulation can extend beyond the mxi-spa locus, we measured the ␤-galactosidase activity of the icsP promoter, which resides outside the mxi-spa locus and also is regulated by VirB, using the plasmid-borne icsP promoter reporter, PicsP-lacZ (pAFW04a [19]). The icsP promoter activity increased 3-fold (P ϭ 0.003) when slyA was exogenously expressed (Fig.…”

“…To measure the SlyA-and VirB-dependent regulation of the promoters controlling mxiC and icsP, ␤-galactosidase activity was measured in cells bearing either the mxiC-lacZ reporter (BS184 [31]) or PicsP-lacZ (pAFW04a [19]) and carrying either pBAD, pBAD-virB, or pBAD-slyA. Overnight cultures were subcultured 1:100 in LB and grown under derepressed (37°C or wild-type background, respectively) or repressed (30°C or virB::Tn5 mutant background, respectively) conditions.…”

“…Encoded on the large virulence plasmid is VirB, a transcriptional regulator essential to Shigella virulence. VirB functions to counteract the repression of virulence gene promoters mediated by H-NS (17)(18)(19), a histone-like nucleoid structuring protein that prevents the inappropriate expression of horizontally acquired genes (reviewed in reference 20). As a derepressor, VirB binds to promoters to facilitate the rearrangement of the H-NS-DNA nucleoprotein complex to ultimately upregulate transcription.…”

Regulatory Hierarchies Controlling Virulence Gene Expression in Shigella flexneri and Vibrio cholerae

“…The icsP promoter is silenced by H-NS and VirB acts there as an anti-repressor; contact between the counteracting proteins may involve DNA dependent protein polymerization in vivo ( Weatherspoon-Griffin et al, 2018 ). The ospZ promoter is also controlled by a VirB/H-NS-dependent silencing/anti-silencing mechanism ( Basta et al, 2013 ). The OspZ protein is secreted by the S. flexneri T3SS to manipulate the host inflammatory response ( Zurawski et al, 2008 ; Zhang et al, 2016 ).…”